High-throughput microbead assay system with a portable, cost-effective Wi-Fi imaging module, and disposable multi-layered microfluidic cartridges for virus and microparticle detection, and tracking.

In recent years biomedical scientific community has been working towards the development of high-throughput devices that allow a reliable, rapid and parallel detection of several strains of virus or microparticles simultaneously. One of the complexities of this problem lies on the rapid prototyping of new devices and wireless rapid detection of small particles and virus alike. By reducing the complexity of microfluidics microfabrication and using economic materials along with makerspace tools (Kundu et al. 2018) it is possible to provide an affordable solution to both the problems of high-throughput devices and detection technologies. We present the development of a wireless, standalone device and disposable microfluidics chips that rapidly generate parallel readouts for selected, possible virus variants from a nasal or saliva sample, based on motorized and non-motorized microbeads detection, and imaging processing of the motion tracks of these beads in micrometers. Microbeads and SARS-CoV-2 COVID-19 Delta variant were tested as proof-of-concept for testing the microfluidic cartridges and wireless imaging module. The Microbead Assay (MA) system kit consists of a Wi-Fi readout module, a microfluidic chip, and a sample collection/processing sub-system. Here, we focus on the fabrication and characterization of the microfluidic chip to multiplex various micrometer-sized beads for economic, disposable, and simultaneous detection of up to six different viruses, microparticles or variants in a single test, and data collection using a commercially available, Wi-Fi-capable, and camera integrated device (Fig. 1).

High-throughput microfluidic droplets in biomolecular analytical system: A review.

Droplet microfluidic technology has revolutionized biomolecular analytical research, as it has the capability to reserve the genotype-to-phenotype linkage and assist for revealing the heterogeneity. Massive and uniform picolitre droplets feature dividing solution to the level that single cell and single molecule in each droplet can be visualized, barcoded, and analyzed. Then, the droplet assays can unfold intensive genomic data, offer high sensitivity, and screen and sort from a large number of combinations or phenotypes. Based on these unique advantages, this review focuses on up-to-date research concerning diverse screening applications utilizing droplet microfluidic technology. The emerging progress of droplet microfluidic technology is first introduced, including efficient and scaling-up in droplets encapsulation, and prevalent batch operations. Then the new implementations of droplet-based digital detection assays and single-cell muti-omics sequencing are briefly examined, along with related applications such as drug susceptibility testing, multiplexing for cancer subtype identification, interactions of virus-to-host, and multimodal and spatiotemporal analysis. Meanwhile, we specialize in droplet-based large-scale combinational screening regarding desired phenotypes, with an emphasis on sorting for immune cells, antibodies, enzymatic properties, and proteins produced by directed evolution methods. Finally, some challenges, deployment and future perspective of droplet microfluidics technology in practice are also discussed.

Implementation of blood-brain barrier on microfluidic chip: Recent advance and future prospects.

The complex structure of the blood-brain barrier (BBB) hinders its modeling and the treatment of brain diseases. The microfluidic technology promotes the development of BBB-on-a-chip platforms, which can be used to reproduce the complex brain microenvironment and physiological reactions. Compared with traditional transwell technology, microfluidic BBB-on-a-chip shows great technical advantages in terms of flexible control of fluid shear stress in the chip and fabrication efficiency of the chip system, which can be enhanced by the development of lithography and three-dimensional (3D) printing. It is convenient to accurately monitor the dynamic changes of biochemical parameters of individual cells in the model by integrating an automatic super-resolution imaging sensing platform. In addition, biomaterials, especially hydrogels and conductive polymers, solve the limitations of microfluidic BBB-on-a-chip by compounding onto microfluidic chip to provide a 3D space and special performance on the microfluidic chip. The microfluidic BBB-on-a-chip promotes the development of basic research, including cell migration, mechanism exploration of neurodegenerative diseases, drug barrier permeability, SARS-CoV-2 pathology. This study summarizes the recent advances, challenges and future prospects of microfluidic BBB-on-a-chip, which can help to promote the development of personalized medicine and drug discovery.

Microfluidic chain reaction of structurally programmed capillary flow events.

Chain reactions, characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (for example, combustion engines), nuclear and biotechnological (for example, polymerase chain reaction) applications1-5. At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling dominoes and Rube Goldberg machines. On the other hand, the microfluidic lab-on-a-chip (also called a micro-total analysis system)6,7 was visualized as an integrated chip, akin to microelectronic integrated circuits, yet in practice remains dependent on cumbersome peripherals, connections and a computer for automation8-11. Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible12-19. Here we introduce the microfluidic chain reaction (MCR) as the conditional, structurally programmed propagation of capillary flow events. Monolithic chips integrating a MCR are three-dimensionally printed, and powered by the free energy of a paper pump, autonomously execute liquid handling algorithms step-by-step. With MCR, we automated (1) the sequential release of 300 aliquots across chained, interconnected chips, (2) a protocol for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibodies detection in saliva and (3) a thrombin generation assay by continuous subsampling and analysis of coagulation-activated plasma with parallel operations including timers, iterative cycles of synchronous flow and stop-flow operations. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ and can form a frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.

Developing a SARS-CoV-2 Antigen Test Using Engineered Affinity Proteins.

The ongoing COVID-19 pandemic has clearly established how vital rapid, widely accessible diagnostic tests are in controlling infectious diseases and how difficult and slow it is to scale existing technologies. Here, we demonstrate the use of the rapid affinity pair identification via directed selection (RAPIDS) method to discover multiple affinity pairs for SARS-CoV-2 nucleocapsid protein (N-protein), a biomarker of COVID-19, from in vitro libraries in 10 weeks. The pair with the highest biomarker sensitivity was then integrated into a 10 min, vertical-flow cellulose paper test. Notably, the as-identified affinity proteins were compatible with a roll-to-roll printing process for large-scale manufacturing of tests. The test achieved 40 and 80 pM limits of detection in 1× phosphate-buffered saline (mock swab) and saliva matrices spiked with cell-culture-generated SARS-CoV-2 viruses and is also capable of detection of N-protein from characterized clinical swab samples. Hence, this work paves the way toward the mass production of cellulose paper-based assays which can address the shortages faced due to dependence on nitrocellulose and current manufacturing techniques. Further, the results reported herein indicate the promise of RAPIDS and engineered binder proteins for the timely and flexible development of clinically relevant diagnostic tests in response to emerging infectious diseases.

A fully automated microfluidic PCR-array system for rapid detection of multiple respiratory tract infection pathogens.

Rapid and accurate identification of respiratory tract infection pathogens is of utmost importance for clinical diagnosis and treatment, as well as prevention of pathogen transmission. To meet this demand, a microfluidic chip-based PCR-array system, Onestart, was developed. The Onestart system uses a microfluidic chip packaged with all the reagents required, and the waste liquid is also collected and stored on the chip. This ready-to-use system can complete the detection of 21 pathogens in a fully integrated manner, with sample lysis, nucleic acid extraction/purification, and real-time PCR sequentially implemented on the same chip. The entire analysis process is completed within 1.5 h, and the system automatically generates a test report. The lower limit-of-detection (LOD) of the Onestart assay was determined to be 1.0 × 103 copies·mL-1. The inter-batch variation of cycle threshold (Ct) values ranged from 0.08% to 0.69%, and the intra-batch variation ranged from 0.9% to 2.66%. Analytical results of the reference sample mix showed a 100% specificity of the Onestart assay. The analysis of batched clinical samples showed consistency of the Onestart assay with real-time PCR. With its ability to provide rapid, sensitive, and specific detection of respiratory tract infection pathogens, application of the Onestart system will facilitate timely clinical management of respiratory tract infections and effective prevention of pathogen transmission. Onestart, a ready-to-use system, can detect 21 pathogens in a fully integrated manner on a microchip within 1.5 h.

Rapid, label-free, and sensitive point-of-care testing of anti-SARS-CoV-2 IgM/IgG using all-fiber Fresnel reflection microfluidic biosensor.

The ongoing global pandemic of SARS-CoV-2 has promoted to develop novel serological testing technologies since they can be effectively complementary to RT-PCR. Here, a new all-fiber Fresnel reflection microfluidic biosensor (FRMB) was constructed through combining all-fiber optical system, microfluidic chip, and multimode fiber bio-probe. The transmission of the incident light and the collection and transmission of Fresnel reflection light are achieved using a single-multi-mode fiber optic coupler (SMFC) without any other optical separation elements. This compact design greatly simplifies the whole system structure and improves light transmission efficiency, which makes it suitable for the label-free, sensitive, and easy-to-use point-of-care testing (POCT) of targets in nanoliter samples. Based on Fresnel reflection mechanism and immunoassay principle, both the SARS-CoV-2 IgM and IgG antibodies against the SARS-CoV-2 spike protein could be sensitively quantified in 7 min using the secondary antibodies-modified multimode fiber bio-probe. The FRMB performs in one-step, is accurate, label-free, and sensitive in situ/on-site detection of SARS-CoV-2 IgM or IgG in serum with simple dilution only. The limits of detection of SARS-CoV-2 IgM and SARS-CoV-2 IgG were 0.82 ng/mL and 0.45 ng/mL, respectively. Based on our proposed theory, the affinity constants of SARS-CoV-2 IgM or IgG antibody and their respective secondary antibodies were also determined. The FRMB can be readily extended as a universal platform for the label-free, rapid, and sensitive in situ/on-site measurement of other biomarkers and the investigation of biomolecular interaction.

Frontline Screening for SARS-CoV-2 Infection at Emergency Department Admission by Third Generation Rapid Antigen Test: Can We Spare RT-qPCR?

To complement RT-qPCR testing for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, many countries have introduced the use of rapid antigen tests. As they generally display lower real-life performances than expected, their correct positioning as frontline screening is still controversial. Despite the lack of data from daily clinical use, third generation microfluidic assays (such as the LumiraDx SARS-CoV-2 Ag test) have recently been suggested to have similar performances to RT-qPCR and have been proposed as alternative diagnostic tools. By analyzing 960 nasopharyngeal swabs from 960 subjects at the emergency department admissions of a tertiary COVID-19 hospital, LumiraDx assay demonstrated a specificity of 97% (95% CI: 96-98), and a sensitivity of 85% (95% CI: 82-89) in comparison with RT-qPCR, which increases to 91% (95% CI: 86-95) for samples with a cycle threshold ≤ 29. Fifty false-negative LumiraDx-results were confirmed by direct quantification of genomic SARS-CoV-2 RNA through droplet-digital PCR (median (IQR) load = 5880 (1657-41,440) copies/mL). Subgenomic N and E RNAs were detected in 52% (n = 26) and 56% (n = 28) of them, respectively, supporting the presence of active viral replication. Overall, the LumiraDx test complies with the minimum performance requirements of the WHO. Yet, the risk of a misrecognition of patients with active COVID-19 persists, and the need for confirmatory RT-qPCR should not be amended.

Optofluidic multiplex detection of single SARS-CoV-2 and influenza A antigens using a novel bright fluorescent probe assay.

The urgency for the development of a sensitive, specific, and rapid point-of-care diagnostic test has deepened during the ongoing COVID-19 pandemic. Here, we introduce an ultrasensitive chip-based antigen test with single protein biomarker sensitivity for the differentiated detection of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A antigens in nasopharyngeal swab samples at diagnostically relevant concentrations. The single-antigen assay is enabled by synthesizing a brightly fluorescent reporter probe, which is incorporated into a bead-based solid-phase extraction assay centered on an antibody sandwich protocol for the capture of target antigens. After optimization of the probe release for detection using ultraviolet light, the full assay is validated with both SARS-CoV-2 and influenza A antigens from clinical nasopharyngeal swab samples (PCR-negative spiked with target antigens). Spectrally multiplexed detection of both targets is implemented by multispot excitation on a multimode interference waveguide platform, and detection at 30 ng/mL with single-antigen sensitivity is reported.

Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples.

The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.

A Highly Automated Mobile Laboratory for On-site Molecular Diagnostics in the COVID-19 Pandemic.

BACKGROUND: Infectious disease outbreaks such as the COVID-19 (coronavirus disease 2019) pandemic call for rapid response and complete screening of the suspected community population to identify potential carriers of pathogens. Central laboratories rely on time-consuming sample collection methods that are rarely available in resource-limited settings. METHODS: We present a highly automated and fully integrated mobile laboratory for fast deployment in response to infectious disease outbreaks. The mobile laboratory was equipped with a 6-axis robot arm for automated oropharyngeal swab specimen collection; virus in the collected specimen was inactivated rapidly using an infrared heating module. Nucleic acid extraction and nested isothermal amplification were performed by a "sample in, answer out" laboratory-on-a-chip system, and the result was automatically reported by the onboard information platform. Each module was evaluated using pseudovirus or clinical samples. RESULTS: The mobile laboratory was stand-alone and self-sustaining and capable of on-site specimen collection, inactivation, analysis, and reporting. The automated sampling robot arm achieved sampling efficiency comparable to manual collection. The collected samples were inactivated in as short as 12 min with efficiency comparable to a water bath without damage to nucleic acid integrity. The limit of detection of the integrated microfluidic nucleic acid analyzer reached 150 copies/mL within 45 min. Clinical evaluation of the onboard microfluidic nucleic acid analyzer demonstrated good consistency with reverse transcription quantitative PCR with a κ coefficient of 0.979. CONCLUSIONS: The mobile laboratory provides a promising solution for fast deployment of medical diagnostic resources at critical junctions of infectious disease outbreaks and facilitates local containment of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) transmission.

Machine learning-based cytokine microarray digital immunoassay analysis.

Serial measurement of a large panel of protein biomarkers near the bedside could provide a promising pathway to transform the critical care of acutely ill patients. However, attaining the combination of high sensitivity and multiplexity with a short assay turnaround poses a formidable technological challenge. Here, the authors develop a rapid, accurate, and highly multiplexed microfluidic digital immunoassay by incorporating machine learning-based autonomous image analysis. The assay has achieved 12-plexed biomarker detection in sample volume <15 µL at concentrations < 5 pg/mL while only requiring a 5-min assay incubation, allowing for all processes from sampling to result to be completed within 40 min. The assay procedure applies both a spatial-spectral microfluidic encoding scheme and an image data analysis algorithm based on machine learning with a convolutional neural network (CNN) for pre-equilibrated single-molecule protein digital counting. This unique approach remarkably reduces errors facing the high-capacity multiplexing of digital immunoassay at low protein concentrations. Longitudinal data obtained for a panel of 12 serum cytokines in human patients receiving chimeric antigen receptor-T (CAR-T) cell therapy reveals the powerful biomarker profiling capability. The assay could also be deployed for near-real-time immune status monitoring of critically ill COVID-19 patients developing cytokine storm syndrome.

PCR past, present and future.

PCR has become one of the most valuable techniques currently used in bioscience, diagnostics and forensic science. Here we review the history of PCR development and the technologies that have evolved from the original PCR method. Currently, there are two main areas of PCR utilization in bioscience: high-throughput PCR systems and microfluidics-based PCR devices for point-of-care (POC) applications. We also discuss the commercialization of these techniques and conclude with a look into their modifications and use in innovative areas of biomedicine. For example, real-time reverse transcription PCR is the gold standard for SARS-CoV-2 diagnoses. It could also be used for POC applications, being a key component of the sample-to-answer system.

Massively multiplexed nucleic acid detection with Cas13.

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples1-3 while simultaneously testing for many pathogens4-6. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents7 self-organize in a microwell array8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health9-11.

Rapid Differential Diagnosis of Seven Human Respiratory Coronaviruses Based on Centrifugal Microfluidic Nucleic Acid Assay.

With the global outbreak of the coronavirus disease 2019 (COVID-19), the highly infective, highly pathogenic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has attracted great attention. Currently, a method to simultaneously diagnose the seven known types human coronaviruses remains lacking and is urgently needed. In this work, we successfully developed a portable microfluidic system for the rapid, accurate, and simultaneous detection of SARS-CoV, middle east respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2, and four other human coronaviruses (HCoVs) including HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. The disk-like microfluidic platform integrated with loop-mediated isothermal amplification provides highly accurate, sensitive, and specific results with a wide linear range within 40 min. The diagnostic tool achieved 100% consistency with the "gold standard" polymerase chain reaction in detecting 54 real clinical samples. The integrated system, with its simplicity, is urgently needed for the diagnosis of SARS-CoV-2 during the COVID-19 pandemic.

Microfluidic Immunoassays for Sensitive and Simultaneous Detection of IgG/IgM/Antigen of SARS-CoV-2 within 15 min.

The outbreak of SARS-CoV-2 is posing serious global public health problems. Facing the emergence of this pandemic, we established a portable microfluidic immunoassay system for easy-to-use, sensitive, rapid (<15 min), multiple, and on-site detection of IgG/IgM/Antigen of SARS-CoV-2 simultaneously. This integrated method was successfully applied for detecting SARS-CoV-2 IgM and IgG antibodies in clinical human serum as well as SARS-CoV-2 antigen in pharyngeal swabs from 26 patients with COVID-19 infection and 28 uninfected people. The assay demonstrated high sensitivity and specificity, which is promising for the diagnosis and monitoring as well as control of SARS-CoV-2 worldwide.